The present invention relates to neuronal formation and methods of treating diseases characterized by abnormalities in and the activity of dopaminergic (DA) and serotonergic (5HT) neurons.
The vertebrate nervous system is composed of multiple neuronal and non-neuronal cell types which develop in stereotypic positions along the dorso-ventral (D-V) and anterior-posterior (A-P) axes of the early neural tube. The mechanisms controlling this process, which is essential for subsequent formation of functional neural networks, are not fully understood (reviewed in Lumsden and KrumIauf, (1996) Science 274: 1109-1114; Tanabe and Jessell, (1996) Science 274: 1115-1123. However, it has been proposed that signaling centers which operate along the two main axes of this system establish an epigenetic grid of Cartesian coordinates, and that neural progenitors assess their location on this grid, assuming distinct cell fates accordingly (e.g. Wolpert, (1969) J. Theor. Biol. 25: 1-47; Rubenstein et al., (1994) Science 266: 578-80.
Consistent with the epigenetic grid hypothesis, grafting experiments have epeatedly demonstrated that vertebrate neural progenitors are not genetically pre-determined, but instead can acquire new stereotypic identities if moved to ectopic locations in the neural plate (Alvarado-Mallartet al., (1990) Develop. Biol. 139: 75-88; Gardner and Barald, (1991) Develop. 113: 1037-1048; Grapin-Botton et al., Develop. 124: 849-859 (1997); Itasaki et al. (1996) Neuron 16: 487-500; Simon et al. (1995) Current Biol. 5: 205-214. In addition, transplantation, as well as explant culture studies have confirmed the existence of signaling centers which can change the fate of juxtaposed neural progenitors. Thus, signaling centers such as the dorsal ectodermal epidermis, roof plate, floor plate and notochord have been shown to instruct cell fates along the D-V axis [reviewed in Tanabe and Jessell (1996) Science 274: 1115-1123; Liem et al. (1997) Cell 91: 127-138] whereas signaling centers located in the prechordal plate, paraxial mesoderm, somitic mesoderm, mid-hindbrain boundary (isthmus) and the anterior neural plate [reviewed in Lumsden and Krumlauf (1996) Science 274: 1109-1114; Dale et al. (1997) Cell 90: 257-269; Foley et al. (1997) Develop. 124: 2983-2996; Grapil-Botton et al. (1997 supra; Muhr et al. (1997) Neuron 19: 487-502; Itasaki et al. (1996) Neuron 16: 487-500; Shimamura and Rubenstein, (1997) Develop. 124: 2709-2718; Houart et al., (1998) Nature 391: 788-792] can change cell fate along the A-P axis of the neural tube. Finally, in agreement with the notion that the information grids are established by extra-cellular molecules, a number of secreted proteins and chemicals were shown to modify the fate of neural plate cells in a characteristic fashion. Thus, Sonic hedgehog and BMP proteins were shown to influence cell fate along the D-V axis (reviewed in Tanabe and Jessell (1996), supra; Liem et al. (1997) Cell 91: 127-138, whereas FGF2, FGF8, retinoic acid and Wnt1 can change cell fate along the A-P axis [reviewed in Lumsden and Krumlauf (1996), supra; Crossley et al. (1996) Nature 380: 66-68; Shimamura and Rubenstein (1997), supra.].
Serotonergic neurons are concentrated in the ventral and ventro-lateral aspects of the hindbrain and innervate most parts of the central nervous system including the cerebral cortex, limbic system and spinal cord. These neurons control levels of awareness, arousal, behavioral traits and food intake, and their abnormal function has been linked to aggression, depression and schizophrenia (Jacobs and Gelperin (1981) Serotonin Neurotransmission and Behavior. The MIT Press, Cambridge, Mass.). Serotonergic neurons important in regulation of food intake, hormone secretion, responses to stress, pain and immune function. Serotonergic neurons innervate nearly every area of the central nervous system, including the cerebral cortex, limbic system and spinal cord, and can influence multiple functions of the brain, such as behavior, appetite, pain, sexual activity, cardiovascular function, hormone secretion, and temperature regulation. Serotonergic dysfunction likely to play roles in the pathophysiology of various psychiatric, neurologic, and other diseases. For example, mental depression, Asberg et al., J. Clin. Psychiatry 47(4): 23-35 (1986), suicide, Asberg et al., supra, Lester, D., Pharmocopsychiatry 28(2): 45-50 (1995), and violent aggressive behavior, Brown et al., J. Clin. Psychiatry 54(4): 31-41 (1990), Eichelman, B. S., Annu. Rev. Med. 41: 149-158 (1990). Serotonin uptake inhibitors have been used in the treatment of mental depression, obsessive-compulsive disorder and bulimia. Fuller, R. W., "Serotonin uptake inhibitors: Uses in clinical therapy and in laboratory research," Progress in Drug Research 45: 167-204, Birkhauser-Varlag, Basel (1995). As serotonergic neurons innervate cerebral blood flow, serotonin receptor agonists, such Sumatriptan, have been employed to abort migraine attacks. Plosker, G. L. et al. Drugs 94(4): 622-651 (1994). Most of the known and cloned serotonin receptors belong to a G-protein-coupled superfamily of receptors having seven membrane-spanning domains. Hoyer et al., Pharmacol. Rev. 46(2): 157-203 (1994). Some seratonin receptor subtypes couple negatively to adenylate cyclase, while others couple positively, while others are coupled to activation of phospholipase C, or ligand-gated ion channels. Fuller, R. W., Ann. N.Y. Acad. Sci. 780: 176-184 (1996).
Dopaminergic (DA) neurons, which reside in the ventral and ventro-lateral aspects of the midbrain, control postural reflexes, movement and reward-associated behaviors. DA neurons develop in the vicinity of the floor plate and are induced by contact-mediation. These neurons innervate multiple structures in the forebrain, and their degeneration or abnormal function is associated with Parkinson's disease, schizophrenia and drug addiction. Hynes et al., Cell 80: 95-101 (1995).
DA neurons located in the substantia nigra have a great impact upon striatal activity as bilateral lesions of the nigrostriatal pathway produce a syndrome in experimental animals that is quite similar to the observed motor dysfunctions observed in Parkinson's disease: resting tremor, rigidity, akinesia and postural abnormalities. Bilateral lesions of the nigrostriatal pathway caused by 6-hydroxydopamine (OHDA) caused profound akinesia, adipsia, aphagia and sensory neglect in rodents, Ungerstedt, U. Acta Physiol. Scand. 1971 (Suppl. 367): 95-121; Yirek and Sladek, 1990, Annu. Rev. Neurosci. 13: 415-440.
Loss of striatal DA is associated with an alternation in the number of target receptors located on striatal cells. In parkinsonism, changes in the status of DA receptors may be dependent on the stage of progression of the disease. The hallmark of parkinsonism is a severe reduction of dopamine in all components of the basal ganglia, Hornykiewicz, O., 1988, Mt. Sinai J. Med. 55: 11-20. Dopamine and its metabolites are depleted in the caudate nucleus, putamen, globus pallidus and pars compacta of the substantia nigra. Moderate losses of DA are found in the nucleus accumbens, lateral hypothalamus, medial olfactory region, and amygdaloid accumbens. Changes in non dopaminergic neuronal systems include decreases in tissue concentrations of norepinephrine, serotonin, substance P, neurotension and several neuropeptides in most basal gangliar structure, cerebellar cortex, and spinal cord.
Considerable attention has been placed on neural transplantation in patients afflicted with Parkinson's disease. These clinical experiments essentially evolved from basic scientific research using various animal models of parkinsonism as recipients of either fetal embryonic nerve cell or paraneuronal tissue grafts to brain-damaged areas. While the concept for neural transplantation is quite old, major advances have occurred only within the last two decades, and many issues remain such as the potential long-term effectiveness of neural grafts to restore and maintain normalized function in animal models of a variety of disorders. Animal experimentation with fetal DA nerve cell grafts have provided encouragement that such grafts could reverse DA deficits and restore motor function in animals with experimental lesions of the nigrostriatal DA system. However numerous ethical, legal and safety issues are coincident with the use of fetal tissue in clinical research, factors which have only exacerbated an already limited supply, all of which establishes an urgent need for alternative sources of dopaminergic neurons.
Schizophrenia is often characterized by peculiar thought disorders, a disturbance of emotional or affective responses to the environment and autism--a withdrawal from interactions with other people. Hallucinations have also been associated as symptomatic of schizophrenia. Phenothiazine drugs are generally acknowledged to be effective in alleviating the symptoms of schizophrenia. Other medications have involved neurotransmitters. Snyder et al., Science 184:1243-1253 (1974). Extended use of and toxic doses of amphetamines also elicit schizophrenic-like symptoms.
Despite the evidence for the existence of positional cues [e.g., Shh, FGF4 and FGF8 are known to be expressed in combination during node and limb bud development. Bueno et al., Develop. Dynam. 207: 291-299 (1996)], there has still been no direct demonstration that two molecularly defined, secreted, physiological signals operate along the A-P and D-V axes respectively, which determine the location and phenotype of a particular neuronal cell type in the neural tube.
Applicants demonstrate herein, that DA neurons in fact, develop at sites where the signals of two distinct molecules, Shh and FGF8, intersect, and that these two extracellular inducers are necessary and sufficient to define the location of DA neurons along the D-V and A-P axes of the anterior neural tube. Progenitors for rostral hindbrain serotonergic neurons appear to use the same intersection as a landmark for specification, but assume a distinct identity because their response to Shh and FGF8 is modified by FGF4.